Citations

Abstract Acquired somatic mutations in ATRX, an X-linked gene encoding a chromatin-associated protein, were recently identified in 4 patients with the rare subtype of myelodysplastic syndrome (MDS) associated with α thalassemia (ATMDS). Here we describe a series of novel point mutations in ATRX detected in archival DNA samples from marrow and/or blood of patients with ATMDS by use of denaturing high-performance liquid chromatography (DHPLC), a technique sensitive to low-level mosaicism. Two of the new mutations result in changes in amino acids altered in previously described pedigrees with germ line ATRX mutations (ATR-X syndrome), but the hematologic abnormalities were much more severe in the patients with ATMDS than in the corresponding constitutional cases. In one ATMDS case where DNA samples from several time points were available, the proportion of ATRX-mutant subclones correlated with changes in the amount of hemoglobin H. This study strengthens the link between acquired, somatic ATRX mutations and ATMDS, illustrates how molecular defects associated with MDS and other hematologic malignancies masked by somatic mosaicism may be detected by DHPLC, and shows that additional factors increase the severity of the hematologic phenotype of ATRX mutations in ATMDS.

Carbon monoxide (CO) is proposed as a physiological messenger. CO activates cGMP and has a direct effect on potassium channels. Both actions of CO lead to hyperpolarization of a cell's resting membrane potential, suggesting that CO may function as a hyperpolarizing factor, although direct evidence is still lacking. Here we take advantage of the known membrane potential gradient that exists in the muscle layers of the gastrointestinal tract to determine whether CO is an endogenous hyperpolarizing factor. We find that heme oxygenase-2-null mice have depolarized smooth muscle cells and that the membrane potential gradient in the gut is abolished. Exogenous CO hyperpolarizes the membrane potential. Regions of the canine gastrointestinal tract that are more hyperpolarized generate more CO and have higher heme oxygenase activity than more depolarized regions. Our results suggest that CO is a critical hyperpolarizing factor required for the maintenance of intestinal smooth muscle membrane potential and gradient.

Background— Recent studies in animals suggest that circulating recipient endothelial precursors may participate in the biology of transplant vasculopathy. It is currently unknown whether a similar interaction between recipient endothelial cells and the vessel wall occurs in human subjects undergoing allogeneic cardiac transplantation. Methods and Results— Circulating endothelial cells and endothelial progenitor cells (EPCs) were quantified in 15 cardiac transplantation subjects with and without angiographic evidence of vasculopathy. In a separate series of experiments, the origin (donor or recipient) of transplant plaque endothelial cells was assessed in subjects who had undergone a gender-mismatched cardiac transplantation and had histological evidence of severe vasculopathy at the time of heart explantation. Circulating EPC outgrowth colonies in peripheral blood were significantly reduced in subjects with transplant vasculopathy compared with those without angiographic evidence of disease (EPC colony-forming units [CFU EPC ]: 4.5±1.9 versus 15.1±3.7, P <0.05). There was no significant difference in circulating endothelial cell numbers as defined by day 4 culture acetylated LDL/lectin assay in either of these patient groups. In a separate group of 5 subjects who underwent gender-mismatched cardiac transplantation, there was a significant seeding of recipient endothelial cells (range: 1% to 24% of all luminal endothelial cells) in large-vessel lumen and adventitial microvessel lumen of arteriopathic vessels. No opposite-sex chimeric cells were observed in control gender-matched transplantation scenarios. Conclusions— These data suggest that the human cardiac transplant arteriopathy is associated with reduction in circulating endothelial precursors and with seeding of recipient-derived endothelial cells at the site of plaque development.

Factors that elevate amyloid-β (Aβ) peptide levels are associated with an increased risk for Alzheimer's disease. Insulysin has been identified as one of several proteases potentially involved in Aβ degradation based on its hydrolysis of Aβ peptides in vitro . In this study, in vivo levels of brain Aβ40 and Aβ42 peptides were found to be increased significantly (1.6- and 1.4-fold, respectively) in an insulysin-deficient gene-trap mouse model. A 6-fold increase in the level of the γ-secretase-generated C-terminal fragment of the Aβ precursor protein in the insulysin-deficient mouse also was found. In mice heterozygous for the insulysin gene trap, in which insulysin activity levels were decreased ≈50%, brain Aβ peptides were increased to levels intermediate between those in wild-type mice and homozygous insulysin gene-trap mice that had no detectable insulysin activity. These findings indicate that there is an inverse correlation between in vivo insulysin activity levels and brain Aβ peptide levels and suggest that modulation of insulysin activity may alter the risk for Alzheimer's disease.

Background and Purpose— Association of mitral valve prolapse (MVP) with ischemic neurological events (INEs) is uncertain. Methods— In the community of Olmsted County (Minn), we identified all MVP diagnosed (1989 to 1998) in patients in sinus rhythm with no prior history of INE. We measured INE rates and compared them with expected rates in our community to define the excess risk of INE. Results— Among 777 eligible subjects (age, 49±20 years; 66% female; follow-up, 5.5±3.0 years), 30 patients had at least 1 INE during follow-up (at 10 years, 7±1%). Compared with expected INEs in the same community, subjects with MVP showed excess risk of lifetime INE (relative risk [RR], 2.2; 95% CI, 1.5 to 3.2; P <0.001) and during follow-up under purely medical management (RR, 1.8; 95% CI, 1.1 to 2.8; P =0.009). Independent determinants of INE were older age (RR, 1.08 per year; 95% CI, 1.04 to 1.11; P <0.001), mitral thickening (RR, 3.2; 95% CI, 1.4 to 7.4; P =0.008), atrial fibrillation (AFib) during follow-up (RR, 4.3; 95% CI, 1.9 to 10.0; P <0.001), and need for cardiac surgery (RR, 2.5; 95% CI, 1.1 to 5.8; P =0.03). INE 10-year rates were low in patients <50 years of age (0.4±0.4%, P =0.60 versus expected) but were excessive in patients >50 years of age (16±3%, P <0.001 versus expected) or with thickened leaflets (7±2%, P <0.001 versus expected). Predictors of follow-up AFib were age, mitral regurgitation, and left atrium diameter (all P <0.01). Conclusions— In the community, subjects with MVP display a lifetime excess rate of INE compared with expected. Clinical (older age) and echocardiographic (leaflets thickening) characteristics define patients with MVP at high risk for INE, and subsequent AFib or need for cardiac surgery, both related to the degree of mitral regurgitation, increase the risk of INE.

Intramembranous proteolysis (IP) is a recently recognized mechanism for transmembrane signal transduction that involves proteolysis of transmembrane proteins within their membrane-spanning domains. Juxtamembranous proteolysis (JP) is similar, but proteolytic cleavage of a transmembrane protein occurs at a site close to, but not within, the transmembrane domain of the target protein. In both IP and JP, a soluble domain of a transmembrane protein is released from its membrane tether. This domain can then transmit a signal either locally or at some distance from the site of cleavage. In certain signaling pathways, JP and IP are linked. JP on one side of the membrane results in secondary IP, which then releases a signaling domain from the membrane. Whereas well-characterized proteases such as caspases, the proteasome, and metalloprotease disintegrins, have been implicated in JP, three families of multipass membrane proteases (MpMPs) have now been shown to carry out IP. Recent studies of events mediated by IP and JP indicate that they regulate key cellular signaling events including pathways involved in sterol regulation, cell fate selection, and growth regulation. Moreover, IP and JP have important roles in certain diseases such as Alzheimer's disease. Because some of the proteases mediating IP and JP can be selectivity inhibited, inhibitors targeting these proteases are likely to alter both physiologic and pathologic events triggered by IP and JP.

Abstract Humans with type-2 diabetes mellitus (TTDM) have hyperglycemia (∼11 mm) and impaired glucose-mediated insulin secretion characterized by impaired first-phase insulin release (FPIR) and pulsatile insulin release. Culture of islets from nondiabetic humans in very high glucose concentrations (∼20–30 mm) for 96 h causes impaired FPIR. We sought to determine 1) whether human islets cultured at a glucose concentration of approximately 11 mm (comparable to TTDM) recapitulates impaired insulin secretion in TTDM, specifically impaired FPIR and insulin pulse mass with an increased proinsulin/insulin (PI/I) secretion ratio; and 2) whether these changes can be attenuated by addition of diazoxide to islets cultured with 11 mm glucose. Islets cultured with 11 mm glucose for 96 h had 75% depleted insulin stores (P &lt; 0.05), decreased FPIR and insulin pulse mass (P &lt; 0.05), and an approximately 3-fold increase in the ratio of PI/I islet content and in secretion ratio (P &lt; 0.05). Addition of diazoxide to islets cultured with 11 mm glucose decreased insulin secretion during static incubation, leading to relative preservation of insulin stores and enhanced insulin secretion during subsequent perifusion; FPIR increased by 162% (P &lt; 0.05) and insulin pulse mass by 150% (P &lt; 0.05) vs. no diazoxide. The mean islet PI/I content and islet PI/I secretion ratio were also decreased by approximately 70% (P &lt; 0.05) by prior addition of diazoxide to islets during culture with 11 mm glucose. FPIR and insulin pulse mass were related to islet insulin stores (P &lt; 0.001 for FPIR and P &lt; 0.001 for pulse amplitude). In conclusion, the pattern of defects of insulin secretion present in TTDM (impaired FPIR and pulsatile insulin secretion, increased PI/I ratio) can be recapitulated in human islets cultured with 11 mm glucose for 96 h. These defects can be at least partially offset by concurrent inhibition of insulin secretion by diazoxide, which also preserves insulin stores. Defective insulin secretion in TTDM may be, at least in part, due to depletion of available insulin stores secondary to chronic increased demand (insulin resistance and hyperglycemia) in the setting of a decreased β-cell mass.

Two substrates of insulin-degrading enzyme (IDE), amyloid β-protein (Aβ) and insulin, are critically important in the pathogenesis of Alzheimer's disease (AD) and type 2 diabetes mellitus (DM2), respectively. We previously identified IDE as a principal regulator of Aβ levels in neuronal and microglial cells. A small chromosomal region containing a mutant IDE allele has been associated with hyperinsulinemia and glucose intolerance in a rat model of DM2. Human genetic studies have implicated the IDE region of chromosome 10 in both AD and DM2. To establish whether IDE hypofunction decreases Aβ and insulin degradation in vivo and chronically increases their levels, we characterized mice with homozygous deletions of the IDE gene (IDE −/−). IDE deficiency resulted in a >50% decrease in Aβ degradation in both brain membrane fractions and primary neuronal cultures and a similar deficit in insulin degradation in liver. The IDE −/− mice showed increased cerebral accumulation of endogenous Aβ, a hallmark of AD, and had hyperinsulinemia and glucose intolerance, hallmarks of DM2. Moreover, the mice had elevated levels of the intracellular signaling domain of the β-amyloid precursor protein, which was recently found to be degraded by IDE in vitro . Together with emerging genetic evidence, our in vivo findings suggest that IDE hypofunction may underlie or contribute to some forms of AD and DM2 and provide a mechanism for the recently recognized association among hyperinsulinemia, diabetes, and AD.